De Smedt H, Parys J B, Wuytack F, Borghgraef R
Biochim Biophys Acta. 1984 Sep 19;776(1):122-32. doi: 10.1016/0005-2736(84)90258-x.
Calcium-induced phosphorylated intermediates and calmodulin-binding proteins in membrane preparations from the renal cortex were analyzed by SDS-polyacrylamide gel electrophoresis at low pH, protein electroblotting and [125I]calmodulin overlay. Two calcium-induced phosphoproteins were found, with a molecular mass of 135 and 115 kDa, respectively. By comparing different preparations characterized by marker enzymes, it was shown that the 135 kDa phosphoprotein is localized in the basal-lateral fragment of the plasma membrane, whereas the 115 kDa phosphoprotein is more pronounced in preparations containing a high proportion of endoplasmic reticulum. A prominent calmodulin-binding protein comigrated with the 135 kDa phosphoprotein; there was no calmodulin binding to polypeptides in the molecular mass range of the 115 kDa phosphoprotein. Partial proteolysis by trypsin and the effect of 20 microM La2+ on the formation of phosphoproteins before and after trypsinization support the conclusion that the 135 kDa protein can be identified with the plasma membrane calcium pump, whereas the 115 kDa phosphoprotein is the phosphorylated intermediate of a different type of calcium pump probably originating from the endoplasmic reticulum. Calmodulin binding in renal membrane preparations analyzed on Laemmli-type slab gels revealed that there are many calmodulin-binding proteins in our preparations. We have identified one band with the renal calcium pump localized in the basal-lateral membrane. Another calmodulin-binding protein migrating at 108 kDa, is not localized in the basal-lateral membrane and could be one of the calmodulin-binding proteins originating from the cytoskeleton.
通过在低pH条件下的SDS-聚丙烯酰胺凝胶电泳、蛋白质电印迹和[125I]钙调蛋白覆盖法，分析了肾皮质膜制剂中钙诱导的磷酸化中间体和钙调蛋白结合蛋白。发现了两种钙诱导的磷蛋白，分子量分别为135 kDa和115 kDa。通过比较以标记酶为特征的不同制剂，结果表明135 kDa的磷蛋白定位于质膜的基底外侧片段，而115 kDa的磷蛋白在含有高比例内质网的制剂中更为明显。一种突出的钙调蛋白结合蛋白与135 kDa的磷蛋白迁移率相同；在115 kDa磷蛋白分子量范围内的多肽没有钙调蛋白结合。胰蛋白酶的部分蛋白水解以及20 microM La2+对胰蛋白酶消化前后磷蛋白形成的影响支持了以下结论：135 kDa的蛋白可被鉴定为质膜钙泵，而115 kDa的磷蛋白是可能起源于内质网的另一种钙泵的磷酸化中间体。在Laemmli型平板凝胶上分析的肾膜制剂中的钙调蛋白结合显示，我们的制剂中有许多钙调蛋白结合蛋白。我们已经鉴定出一条带与定位于基底外侧膜的肾钙泵相关。另一种迁移率为108 kDa的钙调蛋白结合蛋白，不定位于基底外侧膜，可能是起源于细胞骨架的钙调蛋白结合蛋白之一。
